Functional genetic characterization of stress tolerance and biofilm formation in Nakaseomyces (Candida) glabrata via a novel CRISPR activation system

ABSTRACT The overexpression of genes frequently arises in Nakaseomyces (formerly Candida) glabrata via gain-of-function mutations, gene duplication, or aneuploidies, with important consequences on pathogenesis traits and antifungal drug resistance. This highlights the need to develop specific genetic tools to mimic and study genetic amplification in this important fungal pathogen. Here, we report the development, validation, and applications of the first clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system in N. glabrata for targeted genetic overexpression. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in N. glabrata, and further assess optimal guide RNA targeting for robust overexpression. We demonstrate the applications of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance and detect corresponding phenotypic alterations in these key traits, including the characterization of novel phenotypes. Finally, we capture strain variation using our CRISPRa system in two commonly used N. glabrata genetic backgrounds. Together, this tool will expand our capacity for functional genetic overexpression in this pathogen, with numerous possibilities for future applications. IMPORTANCE Nakaseomyces (formerly Candida) glabrata is an important fungal pathogen that is now the second leading cause of candidiasis infections. A common strategy that this pathogen employs to resist antifungal treatment is through the upregulation of gene expression, but we have limited tools available to study this phenomenon. Here, we develop, optimize, and apply the use of CRISPRa as a means to overexpress genes in N. glabrata. We demonstrate the utility of this system to overexpress key genes involved in antifungal susceptibility, stress tolerance, and biofilm growth. This tool will be an important contribution to our ability to study the biology of this important fungal pathogen.

F ungal infections are a significant and growing threat to human health, posing a unique clinical challenge as a result of severely limited diversity of antifungal drugs, and therefore a scarcity of treatment options once drug-resistant infections are diagnosed.Recently, the World Health Organization (WHO) developed the first fungal priority pathogen list (1), highlighting the critical impact of fungal disease on human health and emphasizing antifungal resistance as a "top priority" (1).Among the WHO high-priority pathogens is Nakaseomyces (formerly Candida) glabrata, an opportunistic yeast pathogen of emerging concern.N. glabrata is the second most common cause of candidiasis infections, accounting for ~15% to 25% of invasive infections (2)(3)(4).While Candida albicans remains the most common cause of candidiasis, N. glabrata spp., along with other "non-Candida albicans" species, have been increasing in overall preva lence due in part to reduced susceptibility to antifungal drugs.Indeed, ~20% to 30% of N. glabrata isolates are resistant to azole antifungals, especially fluconazole (5)(6)(7)(8)(9)(10), and resistance to the echinocandin antifungals has increased significantly (from 5% to 12% of isolates) in the past decades (4,9) with ~15% of fluconazole-resistant isolates exhibiting multi-drug resistance (4).Thus, N. glabrata is a critical pathogen with limited therapeutic options requiring focused research efforts to characterize its pathogenicity and mechanisms of antifungal resistance.
While numerous molecular mechanisms are involved in N. glabrata's ability to resist treatment with antifungal agents, a common strategy involves the overexpression of factors involved in enabling growth in the presence of these drugs.Overexpression of genes encoding drug efflux pumps belonging to both the ATP-binding cassette (ABC) superfamily (including CDR1 and CDR2) and the major facilitator superfamily classes is a common strategy to enable N. glabrata to remove antifungals from the cell (4,7,11).Antifungal-resistant N. glabrata strains are commonly identified with gain-of-function mutations in the transactional regulator Pdr1, which promotes overexpression of genes encoding ABC transporters (7,(12)(13)(14)(15)(16)(17).Further, ERG11, which encodes the target of the azole antifungals, is found to be upregulated upon azole treatment in N. glabrata and other Candida species (18), and the ERG11 gene has been found in increased copy number in resistant isolates due to duplication of the ERG11-containing chromosome (19)(20)(21).In addition to an important role in antifungal drug resistance, gene overexpres sion or amplification in N. glabrata is also associated with changes in virulence and interactions with the environment and host species (22)(23)(24), highlighting the critical role of genetic overexpression in N. glabrata adaptation.
This critical role for genetic overexpression in numerous facets of N. glabrata biology demands the development of tools to functionally characterize the overexpression of genes in this important pathogen.Currently, there are several genetic strategies for overexpression in N. glabrata, including promoter replacement strategies to drive high levels of expression (25,26) and engineered expression plasmids (27).These techni ques have provided important advances for the study of N. glabrata and have success fully probed the function of transporter genes involved in antifungal drug resistance.However, there are also limitations to the existing systems, which may rely on poorly efficient homologous recombination for integration, or time-consuming cloning or expensive synthesis of large plasmid constructs.Thus, new techniques, including ones developed in other model systems (28) or well-studied Candida species (29), may be applied in N. glabrata to bolster the currently available genetic tools and expand our capacity for functional gene overexpression in this pathogen.
One advance that has significantly facilitated our capacity for genetic manipulation across a breadth of fungal species (30)(31)(32)(33)(34) is CRISPR-Cas-based techniques.CRISPR activation (CRISPRa) is one such strategy which relies on an endonuclease-dead Cas protein (e.g., dCas9) fused to transcriptional activators.This dCas9-activator complex can be targeted to the promoter region of a target gene via CRISPR single-guide RNAs (sgRNAs) in order to achieve efficient gene activation/overexpression from a gene of interest.CRISPRa has been applied widely in mammalian cell lines, Saccharo myces cerevisiae, and other model systems, and has exploited a diversity of transcrip tional activators to achieve robust gene overexpression (35)(36)(37)(38).More recently, CRISPRa techniques have also been applied in fungal pathogens and other fungal species.In C. albicans, CRISPRa techniques have been successfully applied to drive high levels of expression of genes involved in biofilm formation, antifungal drug resistance, and stress tolerance, and phenotypically validate their role in these processes (39)(40)(41).CRISPRa techniques have also been applied in diverse filamentous fungi for the activation of biosynthetic gene clusters and enhanced production of key bioactive metabolites (42)(43)(44)(45).While CRISPR techniques have been adapted for diverse applications in N. glabrata (30,(46)(47)(48), including a recent CRISPR interference technique using similar principles to CRISPRa for gene repression (49), to date, no CRISPRa systems have been developed for N. glabrata.
Here, we demonstrate the development and first applications of a CRISPRa genetic overexpression system for the fungal pathogen N. glabrata.This episomal plasmid-based CRISPRa system exploits a nuclease-dead dCas9 fused to a tripartite activator complex VP64-p65-Rta (VPR) and a Gibson assembly cloning method for highly efficient single guide RNA integration.We show that this system can drive high levels of genetic overexpression and recapitulate associated antifungal drug resistance and biofilm growth phenotypes.We describe optimal guide RNA targeting rules for robust overex pression in N. glabrata and characterize novel phenotypes involved in stress and drug resistance associated with overexpression.Finally, we capture strain variation using our CRISPRa system in two commonly used N. glabrata genetic backgrounds.Together, this tool will expand our capacity for functional genetic overexpression in this pathogen with numerous possibilities of applications.

Development of a single CRISPRa plasmid in N. glabrata
In order to develop a CRISPRa system for genetic overexpression in N. glabrata, we designed and constructed a single-plasmid CRISPRa plasmid optimized for targeted gene activation in this fungal organism (Fig. 1a).The system relies on a unique self-rep licating and curable plasmid which allows the reversible overexpression of any gene without the need for stable genome modification.In this system, the genome-targeting portion of the guide RNA, the CRISPR RNA (crRNA) sequences, is cloned under the control of the RNA polymerase III SNR52p promoter at the NotI-restriction site, which has been previously validated as a facile system for highly efficient Gibson assembly-based cloning of sgRNA targeting sequences (50).In the same plasmid, we cloned the dCAS9 gene under the control of the strong and constitutive N. glabrata PDC1 promoter (51) and fused dCAS9 to the tri-domain VPR activator complex developed in S. cerevisiae (52).The VPR activator complex is composed of two SV40-NLS sequences, a VP64 domain (composed of four repeats of the minimal activation domain of herpes simplex virus VP16), a SV40-NLS sequence, a linker, a p65 domain (transcriptional activation domain of human RelA), and an Rta AD domain (transcriptional activation domain from the human herpesvirus 4 (Epstein-Barr virus) replication and transcription activator Rta/ BRLF1).The VPR activator complex fused to dCAS9 has previously been demonstrated to robustly activate gene expression in both S. cerevisiae and C. albicans (39,52).This newly developed N. glabrata CRISPRa plasmid has been deposited to Addgene (Table S1, Addgene plasmid catalog #213041).
To confirm that the dCas9-VPR construct did not impact N. glabrata growth, we compared the growth of a strain transformed with the backbone plasmid, pCU-PDC1, from which the CRISPRa plasmid was constructed, with a strain transformed with the CRISPRa plasmid encoding a non-targeting sgRNA.We found no significant impact of the dCas9-VPR construct on N. glabrata's growth (Fig. 1b), suggesting the CRISPRa plasmid does not significantly impact fitness under the conditions tested.
Yeast episomal plasmids have the advantage of being curable from cells by removing the selection pressure, making any system based on these vectors fully reversible.Since our system relies on the URA3 selection marker, cells that have lost the plasmid should be easily selectable after culturing in a non-selective medium (i.e., in YPD).To demonstrate that our CRISPRa plasmid can be cured from N. glabrata cells, we cultured a strain transformed with a non-targeting CRISPRa and a strain transformed with the CRISPRa plasmid overexpressing PDR1.We found that our CRISPRa plasmid can be easily cured from cells after two rounds of culture in YPD with a successful rate of ~14% to 16%, and that whether or not the system is actively targeting a gene does not impact this curability (Fig. 1c and Fig. 1d).

Overexpressing a gene implicated in fluconazole resistance
To validate that this CRISPRa system could effectively overexpress genes in N. glabrata, we first targeted a gene with a well-characterized antifungal drug resistance phenotype associated with high levels of expression.We focused on PDR1, encoding a transcrip tional regulator of a pleiotropic drug resistance network in N. glabrata, whose overex pression is known to play a role in resistance to azole antifungals in clinical isolates (16,(53)(54)(55).Since previous research reported different transcriptional start sites (TSSs) for N. glabrata's PDR1 gene (56,57), we decided to design and clone 10 unique sgRNAs for our CRISPRa plasmid, spanning the promoter region of PDR1 from −736 to +36 bp (relative to the start codon) on both sense and antisense DNA strands.We then determined the susceptibility of these CRISPRa strains in fluconazole using a minimum inhibitory concentration (MIC) assay (Fig. 2a).We were able to see a decrease in fluconazole susceptibility (MIC50 shifted from 80 to 160 µg/mL) with sgRNAs located at −553 and −550 bp, on the sense and antisense strands, respectively.
To further validate the decreased susceptibility in our CRISPRa strains, we performed growth curve assays in medium containing 80 µg/mL of fluconazole and calculated the relative area under the curve.As a control, strains were also cultured in media without drugs (Fig. 2b).No fitness differences between strains were observed when strains were  cultured without drugs; however, in fluconazole, the two CRISPRa strains encoding sgRNAs located at −553 and −550 bp were significantly increased in their ability to grow compared to the non-targeting CRISPRa strain.
To confirm if phenotypic changes in drug susceptibility were due to transcriptional overexpression of PDR1, we quantified the expression of PDR1 in four different CRISPRa strains via real-time quantitative PCR (RT-qPCR) compared with the control strain encoding a non-targeting sgRNA (Fig. 2c).We found that our CRISPRa system enhances PDR1 transcription by ~1.5-fold and that this was achieved by using sgRNAs targeting both the sense or antisense DNA strands.Together, this indicates that our CRISPRa system can drive overexpression of genes in N. glabrata and recapitulate phenotypes associated with genetic overexpression.

Overexpressing a gene implicated in biofilm formation
To demonstrate that our CRISPRa system can be used to assess genes involved in fungal pathogenesis traits, we decided to overexpress EFG1, encoding a transcription factor implicated in the control of biofilm formation (58).This gene has been characterized in N. glabrata and is known to lead to enhanced biofilm growth when overexpressed (58).Since the TSS of EFG1 is well defined, we were able to design 10 sgRNAs to span the promoter of EFG1.We generated 10 N. glabrata strains each containing unique sgRNAs located -462 to +112 bp relative to the TSS and measured the resultant biofilm growth of these strains using XTT-reduction biofilm assays.Three of these CRISPRa strains (encoding sgRNAs located at −190, −105, and −36 bp) significantly have a better ability to form biofilms as compared to the non-targeting CRISPRa strain (Fig. 3a).
We next profiled the expression of EFG1 in our CRISPRa strains and found that EFG1 was overexpressed from ~3.6-to 8.1-fold using our dCas9-VPR construct, with the highest level of expression obtained when targeting the dCas9-VPR construct −105 bp upstream the TSS of EFG1 (Fig. 3b).Compellingly, we found a significant correlation between the level of EFG1 overexpression and the biofilm-forming capacity of the CRISPRa strains (Fig. 3c).We further found that targeting the CDS leads to repression of EFG1, which may be a result of dCas9-VPR sterically interfering with the RNA polymerase II (Fig. 3b and c).

Characterization of two new genes whose overexpression leads to decreased caspofungin susceptibility and better stress tolerance
Finally, we wanted to demonstrate that our CRISPRa system could be exploited to characterize new genes whose overexpression has not previously been profiled.For this, we targeted N. glabrata STE11 and SLT2 genes for overexpression.STE11 encodes a mitogen-activated protein kinase and has been shown to be involved in control of stress response and virulence in N. glabrata (59).Notably, Ste11 has been shown to mediate cross-tolerance to different environmental stresses such as low pH or oxidative stress (60).In the same study, authors stipulated that the identified mutations in STE11 were likely to enhance the activity of Ste11, but the role of STE11's expression levels in stress tolerance has never been studied.SLT2 is also predicted to encode a mitogen-activated protein kinase but with a role in cell wall integrity (61).It has been shown that in N. glabrata, this gene becomes overexpressed following a caspofungin (CASP) treatment (62), but no study has shown that the overexpression of SLT2 is correlated to better tolerance to caspofungin.
We generated CRISPRa strains overexpressing STE11 and SLT2 ~1.2-to 1.8-fold and ~1.8-to 2.8-fold, respectively (Fig. 4).We assessed thermotolerance and tolerance to H 2 O 2 -induced oxidative stress for the STE11-overexpressing CRISPRa strains and caspofungin susceptibility for the SLT2-overexpressing ones.We were able to demon strate that overexpression of STE11 and SLT2 leads to a better tolerance to heat and oxidative stress and to a better fitness in caspofungin, respectively.This demonstrates our ability to exploit this CRISPRa system to investigate novel phenotypes associated with gene overexpression in N. glabrata.The CRISPRa platform is functional in multiple N. glabrata strain backgrounds General physiology differs across different strain backgrounds and, notably, it has been reported how stress sensitivity and growth physiology traits can vary significantly across yeast strains (63,64).In S. cerevisiae, the use of CRISPR technologies across different strain backgrounds can significantly impact phenotypes based on the genetic background (65).A recent study has demonstrated that phenotypic differences, notably in metabolic profiles and cell wall carbohydrate complexity and abundance, exist between the two commonly used N. glabrata isolates, CBS138 and BG2, and that it can influence its pathogenicity (66).All experiments described above in this study were carried out in strain HM100, derived from the reference strain CBS138.To determine possible strain variation responses to our CRISRa system, we decided to test our system in another N. glabrata strain background, BG87 derived from N. glabrata strain BG2.To do so, we overexpressed PDR1 and EFG1 in BG87, employing the same sgRNAs as those used in the HM100 strain.We found that our CRISPRa system works efficiently in strain BG87 but that its portability is influenced by the strain.Indeed, we were able to overexpress PDR1 and EFG1 up to ~1.8-and 5.7-fold, respectively (Fig. 5a); however, of the two sgRNAs leading to PDR1 overexpression in strain HM100, only one increased the transcriptional activity of PDR1 and decreased fluconazole (FLZ) susceptibility in strain BG87 (Fig. 5b and c).Similar results were obtained for EFG1, where only one of three gRNAs that led to strong EFG1 overexpression and a high capacity to form biofilm in strain HM100 increased strain BG87's capacity to form biofilm (Fig. 5d).These results may be due to TSS differences between the two strains.

DISCUSSION
Here, we report the development, validation, and application of the first CRISPRa technology in N. glabrata.Where nearly all CRISPR systems developed in N. glabrata rely on dual plasmid systems (46,49), ours relies on a single centromeric plasmid which limits the need for selectable markers and is not reliant on inefficient homologous recombination in N. glabrata, as no stable genome integration is needed.Since it is a centromeric plasmid, selection pressure must be maintained during experiments in order to maintain plasmid-containing cells.Our CRISPRa system further employs URA3 as a selectable marker, which allows a non-toxic selection pressure and avoids the risk of potential synergistic effects with other drugs used during experimentimentation.In our plasmid system, sgRNAs can easily be cloned at the NotI-restriction site using the highly efficient Gibson assembly method.Finally, since our system is constitutive, it does not require specific inducing conditions and is active as soon as it is transformed into cells which facilitate its use.
We have demonstrated the ability of our CRISPRa system to increase the transcrip tional activity of N. glabrata's genes from ~1.5-to 8.0-fold.We found that designing and testing only ~2 to 3 sgRNAs in regions −360 to −9 bp upstream of the TSS of the target gene on either DNA strand are sufficient to achieve high levels of overexpres sion.The ability to target both the sense and antisense DNA strands by our dCas9-VPR construct increases the number of available PAM sites and thus increases the likelihood of identifying usable sgRNAs.Finally, we find that the level of overexpression achieved can be conveniently regulated based on the targeting location of the sgRNA within the promoter region of the target gene, which effectively makes the system titratable.This has been previously reported in many CRISPRa studies in other organisms (35,39,40,67) and constitutes a powerful feature of this and other CRISPRa technologies.
Interestingly, for two genes, we found that a slight increase in transcriptional activity (~1.46-to 1.50-fold for PDR1 and 1.24-to 1.80-fold for STE11) was enough to lead to detectable phenotypes: decreased susceptibility to fluconazole for PDR1 and better stress tolerance for STE11.This is in contrast to EFG1, where expression needed to be increased over 3.5-fold to promote enhanced biofilm growth.For PDR1, gain-of-func tion mutations/duplications of the PDR1 gene or PDR1-containing chromosome are frequently identified as a mechanism of resistance in fluconazole-evolved laboratory and clinical isolates of N. glabrata (17,53,55,(68)(69)(70)(71).While expression of PDR1 in these strains has not typically been studied, our results suggest that even if mutations lead to a weak increased transcriptional activity (below twofold), it may lead to reduced susceptibility to fluconazole, which clinically represents a threat to treatment success.In addition, we found a strong correlation between the level of overexpression induced by our dCas9-VPR construct and the magnitude of the resulting phenotype.The ability to modulate the levels of expression using diverse sgRNAs allows us to mimic dosedependent phenotypes that are known to exist in Candida species, such as azole drug resistance associated with dose-dependent expression of TAC1 and ERG11 (72).
The validation of our system in two N. glabrata genetic backgrounds enables us to capture strain variation.Some sgRNAs lead to overexpression in one strain and to repression in another, possibly due to differing TSSs between the two strains.It is possible that the dCas9-VPR construct sterically prevents transcription factors from accessing their binding sites, therefore causing repression of the target gene.Using our CRISPRa system, we were also able to characterize new genes for which overexpres sion leads to a decrease in caspofungin susceptibility and better stress responsiveness, including thermotolerance.This underlies the importance of overexpression alteration in drug resistance and stress response in N. glabrata.While these genes have been previously studied, we demonstrate that novel forms of genetic alterations (i.e., deletions versus overexpression) can help characterize phenotypes in new ways, and therefore, we must use all available approaches (e.g., repression, activation, and deletion) to fully capture the role and regulation of genes.This will ultimately help better predict and understand important facets of biology, including virulence and antifungal drug resistance, in an important fungal pathogen like N. glabrata.Altogether, this unique CRISPRa system will bolster the currently available genetic tools and expand our capacity for functional gene overexpression in this pathogen.Applied at a genome scale, this tool could lead to the identification of new genes implicated in drug resistance, virulence, and stress response.This could lead to the identification of potential new drug targets to ultimately combat this opportunistic pathogen.

Strains and growth conditions
Yeast strains, plasmids, and primers used in this study are listed in Tables S1 through S3, respectively.Yeast strains were grown in broth or on plates at 37°C in synthetic complete medium lacking uracil (SC-Ura) for plasmid selection (0.67% yeast nitrogen base without amino acids with ammonium sulfate, 2% glucose, supplemented with adenine and all amino acids except uracil), YPD (2% peptone, 1% yeast extract, and 2% glucose) or RPMI-1640 supplemented with 3.5% morpholinepropanesulfonic acid and 2% glucose.Escherichia coli DH5α cells were cultured at 30°C in broth or on LB agar plates containing 100-µg/mL ampicillin for plasmid selection.

PCR, transformations, and genomic DNA (gDNA) extraction
PCRs were performed using the Q5 High-Fidelity DNA Polymerase for cloning and crRNA N20 sequence amplification or FroggaBio Taq DNA Polymerase for transformant screening.gDNA was extracted using the Thermo Fisher Scientific Invitrogen PureLink Genomic DNA Mini Kit.
Yeast transformations were performed according to the "one-step" lithium acetate transformation protocol, as previously described (73).Bacterial transformations were performed using NEB 5-alpha competent E. coli cells, according to the manufacturer's recommendations.

Plasmid design and cloning
Molecular cloning was performed using the Gibson assembly method (74).The dCas9 backbone plasmid was synthesized by Twist Bioscience (www.twistbioscience.com)from plasmid pCU-MET3 (51).This dCas9 backbone plasmid contains the URA3 selectable marker, an N. glabrata centromeric origin of replication (CEN/ARS sequence), sgRNA cloning site (SNR52 promoter, NotI cloning locus, and sgRNA tail), and dCas9 (D10A mutation in the RuvC catalytic domain and N863A mutation in the HNH catalytic domain) cloned under the control of the MET3 promoter (MET3p).The MET3 promoter was replaced by the strong and constitutive PDC1 promoter (PDC1p) to allow a strong expression of dCas9 (51).The endogenous PDC1p sequence was amplified using primers PDC1-F and PDC1-dCas9-R and gDNA of strain HM100 as a DNA template.Since primers PDC1-F and PDC1-dCas9-R contain 40 bp identical to regions surrounding the MET3p, it enables a replacement of MET3p by PDC1p into the dCas9 backbone plasmid by Gibson assembly.This gives rise to the PDC1-dCas9 plasmid.Into PDC1-dCas9 we fused a gene encoding dCas9 to a VPR three-part domain {2× SV40NLS-VP64 domain (composed of four repeats of the minimal activation domain of herpes simplex virus VP16), 1× SV40 NLS-p65 domain (transcriptional activation domain of human RelA), Rta AD [transcriptional activation domain from the human herpesvirus 4 (Epstein-Barr virus) replication and transcription activator Rta/BRLF1]} synthesized as a gene fragment from Twist Bioscience.The sequence of this gene fragment can be found in Table S4.This gene fragment was cloned into the dCas9 plasmid backbone using Gibson assembly.The final construct (pCGLM2 aka pRS712) was fully sequenced by Plasmidsaurus (www.plasmidsaurus.com/)and is available via Addgene (www.addgene.org/,catalog #213041).
The control backbone plasmid, pCU-PDC1, was constructed from pCU-MET3 ( 51) by replacing the MET3p with PDC1p.The endogenous PDC1p sequence was amplified using primers PDC1-F and PDC1-R and gDNA from N. glabrata strain HM100 as a DNA template.Since primers PDC1-F and PDC1-R contain 40 bp identical to regions surrounding the MET3p, it enables a replacement of MET3p by PDC1p into the pCU-MET3 by Gibson assembly.This gives rise to the pCU-PDC1 plasmid.

sgRNA design and cloning
The sgRNA and crRNA (N20 nucleotide sequence complementary to the target genomic DNA) were designed based on efficiency and predicted specificity via the sgRNA design tool Eukaryotic Pathogen CRISPR gRNA Design Tool [http://grna.ctegd.uga.edu(75)].Sequences and information on crRNA N20 sequences can be found in Table S5.Each crRNA N20 sequence was ordered from Integrated DNA Technologies (www.idtdna.com)with 20-bp overlaps on either side of the region of target complementary and was amplified with extender oligos (primers gRNA-extender-F and gRNA-extender-R) to extend the overlaps from 20 to 40 bp, as previously described (50).They were then cloned at the NotI-restriction site into pCGLM2 using Gibson assembly.
sgRNAs were designed ~−716 to +36 bp upstream of the start codon for PDR1 and ~−463 to −9 bp from the TSS for EFG1, STE11, and SLT2.Start codons and TSSs were obtained from The Candida Genome Database (76).

Growth curves
N. glabrata transformants carrying pCGLM2 were grown overnight in liquid SC-Ura medium at 37°C, 250 rpm, and used to inoculate fresh SC-Ura or SC-Ura containing either 80, 160, or 320 µg/mL of fluconazole or SC-Ura containing 32 ng/mL of caspofungin at an optical density (OD) 600 of 0.05 or 0.005 in a 96-well flat-bottomed plate with a total volume of 200 µL per well topped with 25 µL of mineral oil to avoid evaporation.The starting OD 600 of each strain was kept consistent within the same experiment.Growth was measured at 37°C via OD 600 at 20-min intervals over the course of 24 h using an Infinite 200 PRO microplate reader (Tecan).Plates were shaken orbitally for 1,000 seconds at a 4.5-mM amplitude between growth measurements.

Plasmid curing
CRISPRa strains were cultured overnight in YPD at 37°C, shaking at 250 rpm.The next day, cells were spread onto YPD plates to obtain single colonies and were grown for 48 h at 37°C.These plates were then replica-plated onto both SC-Ura and YPD plates, and after 48 h at 37°C, cells that have lost the plasmid, growing onto YPD but not SC-Ura, were identified.

MIC assays
MIC assays were performed in at least three replicates following the EUCAST E.DEF v.7.3.2 protocol (77), with the following minor modifications.MICs were run in SC-Ura.
Overnight cultures of N. glabrata grown in SC-Ura were diluted to an OD 530 of 0.01 (~2.10 5 cells) in SC-Ura.Cells were mixed into the plates in an equal volume such that the starting OD 530 values of each strain were 0.005 (~0.5.10 5 cells) in SC-Ura.Plates were incubated at 37°C without shaking, and absorbance values at 530 nm were read after 24 h using an Infinite 200 PRO microplate reader (Tecan).The range of fluconazole concentrations tested was 1,280.0-1.25 µg/mL.

Biofilm assays
Biofilm assays were performed as previously described (78), with minor modifications.N. glabrata cells from SC-Ura O/N cultures were diluted to an OD 600 of 0.01 in RPMI in 200 µL in 96-well plates, and cells were grown statically at 37°C for 48 h.After two washing steps in 1× phosphate-buffered saline (PBS) solution and once biofilms were dried, 90 µL of 1 mg/mL tetrazolium salt (XTT) (prepared in 1× PBS and centrifuged to remove sediment prior to use), and 10 µL of 0.32 mg/mL PMS (prepared in water) was added to each well.Plates were incubated statically at 30°C for 2 h to allow biofilms to reduce XTT, measured at 490 nm using an Infinite 200 PRO microplate reader (Tecan), and normalized to the growth of planktonic cells harvested previously.The "relative biofilm formation" was calculated as follows: (OD 490 /OD 600 planktonic cells) teststrain / (OD 490 /OD 600 planktonic cells) control , where the control represents the non-targeting CRISPRa strain.

Stress assays
Stress assays were performed similarly to those previously described (60), with minor modifications.For the hydroxide peroxide (H 2 O 2 ) stress assays, SC-Ura O/N cultures of the CRISPRa strains were diluted to an OD 600 of 1 in a 96-deep well plate in 500 µL of SC-Ura or SC-Ura containing 100 mM of H 2 O 2 .The plate was then incubated for 1 h in a plate-shaking incubator at 37°C, shaking at 800 rpm.After incubation, cells were centrifuged in the 96-deep well plate at 300 g for 10 min to remove the supernatant.Cells were then resuspended in 100 µL of a sterile PBS solution, diluted in 10-fold serial dilutions in a PBS and each dilution was spotted using a 96-pin pinning tool onto SC-Ura.Plates were then photographed after 48 h at 37°C.
For heat shocks, overnight cultures were diluted to an OD 600 of 0.5 in SC-Ura in microcentrifuge tubes.Tubes on a tray were then incubated for 1 h at 37°C or 47°C, statically.After the incubation, they were placed at 4°C for 5 min.Cells were then diluted and spotted on SC-Ura as described above.Plates were photographed after growth for 48 h at 37°C.

RNA extraction and RT-qPCR
To detect differences in gene expression, overnight cultures of N. glabrata grown in SC-Ura were diluted to an OD 600 of 0.05 in fresh SC-Ura (for SLT2 and STE11) or SC-Ura containing 80 or 160 µg/mL of FLZ (for PDR1) or RPMI (for EFG1) and grown to an OD 600 of ~0.6 to 0.8 at 37°C.Cells were pelleted and frozen at −80°C before RNA was extracted.RNAs were extracted using the RNeasy Mini kit from Qiagen, according to the manufacturer's recommendations.
cDNA was synthesized from RNAs as follows: 1,000 µg of RNA was treated for DNA contamination using the Invitrogen TURBO DNA-free Kit from Thermo Fisher Scientific.The supernatant was then incubated for 5 min at 65°C with 4.5 µM of random hexamers (Thermo Fisher Scientific) and 9 µM of a dNTPs mix (Thermo Fisher Scientific).After the reaction, the mixture was put on ice for 1 min.From this cDNA was synthesized using the SuperScript IV Reverse Transcriptase from Thermo Fisher Scientific and was stored at −20°C, as needed, before running RT-qPCRs.
RT-qPCRs were performed using a QuantStudio 3 Real-Time PCR Instrument (Thermo Fisher Scientific) with the SYBR Green-based method.Primers used for RT-qPCR are listed in Table S3.Expression profiling calculations were performed according to the comparative CT method (79).Briefly, expression values for the targeted gene of interest were compared with the housekeeping gene rRNA 18S to obtain a ΔCT value for each strain.The ΔCT values in the experimental CRISPRa strains were then compared with the non-targeting CRISPRa control strain to obtain a ΔΔCT value and a fold difference in the expression of the target gene.

Analysis and statistics
All graphs and statistical analysis were performed using GraphPad Prism v.9.4.1.Before using a parametric test, the normality of the data sets was checked.Normality was checked using the Shapiro-Wilk test.

FIG 1
FIG 1 The Nakaseomyces glabrata CRISPRa system.(a) Plasmid map of N. glabrata CRISPRa plasmid (pCGLM2 aka pRS712).pCGLM2 is a centromeric Ura+ selectable plasmid.The catalytically dead Cas9 (dCas9) is fused to the VPR activator complex, which is composed of the VP64, p65, and Rta AD activator domains.sgRNAs are cloned at the NotI-restriction site using Gibson assembly.Panel created with BioRender.com,not to scale.(b) Growth of the CRISPRa backbone plasmid (no dCas9-VPR or sgRNA construct) compared with growth of a strain containing a non-targeting CRISPRa plasmid.Cells were grown in SC-Ura at 37°C.Growth was monitored by OD reading at 600 nm every 20 min for 24 h.(c and d) Curing of the CRISPRa plasmid from N. glabrata.Cells that have lost the CRISPRa plasmid are selected after two rounds of culture in YPD at 37°C.P value > 0.05, Wilcoxon-Mann-Whitney U test.All experiments were carried out in three independent replicates.ns, not significant; OD, optical density; SC-Ura, synthetic complete medium lacking uracil.

FIG 2
FIG 2 Validation of the N. glabrata CRISPRa system for overexpressing a gene involved in fluconazole resistance.(a) The fitness of CRISPRa strains overexpressing PDR1 was measured using a MIC assay.Results are presented as a heatmap in which the growth in drugs, relative to the growth in no drug, is depicted, with the value of 1 representing the highest fitness.(b) The fitness of CRISPRa strains overexpressing PDR1 was measured using growth curve assays in the absence of drug (no treatment) and in 80 µg/mL of fluconazole.The relative area under the time-versus-OD curve (AUC) was then calculated.Dots represent the mean AUC, and error bars represent the standard deviation.Differences between groups were tested for significance using the Kruskal-Wallis test for the no-treatment condition and one-way analysis of variance for the fluconazole condition.*P < 0.05, **P < 0.01.All experiments were carried out at least three times independently.(c) Fold change expression of PDR1 in CRISPRa strains overexpressing PDR1.Fold change in the expression of PDR1 relative to the housekeeping gene 18S rRNA in both experimental and non-targeting strains was measured by real-time quantitative PCRs.Relative expression was then calculated using the (Continued on next page)

FIG 2 ( 6 (FIG 3
FIG 2 (Continued)non-targeting CRISPRa strain, and the mean fold differences were plotted.Error bars depict the standard deviation.The dashed line represents the normalized baseline expression of the control strain.The fold change differences were tested for significance using Student's t-test.*P < 0.05, **P < 0.01.All experiments were carried out in at least three independent replicates.AUC, area under the curve; ns, not significant.

FIG 3 (FIG 4
FIG 3 (Continued) (c) The square Pearson's correlation coefficient between the biofilm capacity and the fold change in EFG1 expression in the CRISPRa strains overexpressing EFG1.ns, not significant.

FIG 5
FIG5 Validation of the N. glabrata CRISPRa system in strain BG87.(a) Fold change expression of PDR1 and EFG1 in CRISPRa strains overexpressing PDR1 and EFG1, respectively.Fold change in the expression of PDR1 and EFG1 relative to the housekeeping gene 18S rRNA in both experimental and non-targeting strains was measured by RT-qPCR.Relative expression was then calculated using the non-targeting CRISPRa strain, and the mean fold differences were plotted.Error bars depict the standard deviation.The fold change differences were tested for significance using Student's t-test: *P < 0.05, ****P < 0.0001.All experiments were carried out in at least three independent replicates.(b) The fitness of CRISPRa strains overexpressing PDR1 was measured using MIC assays.Results are presented as a heatmap in which the growth in drugs, relative to the growth in no drug, is depicted, with the value 1 representing the highest fitness.(c) The fitness of CRISPRa strains overexpressing PDR1 was measured using growth curve assays in the absence of drug (no treatment) and in 160 or 320 µg/mL of fluconazole.The relative area under the time-versus-OD curve (AUC) was then calculated.Dots represent the mean AUC, and errors bars represent the standard deviation.Differences between groups were tested for significance using one-way analysis of variance.**P < 0.01.All experiments were carried out in at least three independent replicates.(d) The biofilm-forming capacity of CRISPRa strains overexpressing EFG1 was measured using an XTT-reduction biofilm assay.The relative biofilm formation of CRISPRa strains overexpressing EFG1 was calculated using the non-targeting strain as a control, and the relative log 2 values were calculated.Long dashes on violin plots represent the median, and short dashes are the first and third quartiles.Data were combined from three independent experiments where n = 21.Differences between groups were tested for significance using a one-sample Student's t-test.****P < 0.0001.ns, not significant.